The ability to efficiently and reproducibly generate differentiated cell types from pluripotent stem cells in vitro has opened the door for the development of cell-based therapies for the treatment of a broad range of degenerative and debilitating diseases. Osteoarthritis (OA) is a candidate for such therapy as it affects at least one in ten adults (Lawrence, Felson et al. 2008), leaving patients with a poor quality of life due to pain associated with joint movement. Pathogenic hallmarks of OA include the degradation of the extracellular matrix (ECM) of articular cartilage that lines the joints together with thickening of the underlying subchondral bone and the formation of osteophytes (bone spurs). Articular cartilage is generated by a distinct subpopulation of chondrocytes known as articular chondrocytes (ACs) that are specified early in development and persist throughout adult life. While ACs function to maintain integrity of the articular cartilage under normal circumstances, they display little capacity to repair cartilage damaged by injury or disease. Consequently, with disease progression, damage to the cartilage is so extensive that surgical intervention such as joint replacement is often required to improve the quality of life for the patient. ACs differ from growth plate chondrocytes (GPCs), whose primary function is to form bone through the process of endochondral ossification (Colnot, 2005). Interestingly, with the onset of OA, ACs appear to acquire some characteristics of GPCs, including hypertrophy, which may contribute to the pathogenesis of this disease.
Chondrocyte and cartilage replacement represent a potential new therapy for OA that could, at some point dramatically reduce the need for mechanical devices. This type of therapy, however, is dependent on access to appropriate tissue and sufficient numbers of highly enriched ACs. It is well established that adult mesenchymal stem cells (MSCs) are able to differentiate to chondrocytes in vitro, however, it is unclear if they are able to give rise to ACs as the cartilage-like tissue generated from them prematurely undergoes hypertrophy (Pelttari, Winter et al. 2006, Steinert, Ghivizzani et al. 2007, Pelttari, Steck et al. 2008) Alternatively, ACs have been harvested directly from patients and used for tissue generation ex vivo, despite their limited capacity to proliferate. Tissue generated by passaged chondrocytes exhibits fibrocartilage characteristics, which can improve the quality of life for the patient in the short term but ultimately undergoes degradation as it lacks sufficient weight bearing capacity (Tins, McCall et al. 2005, LaPrade, Bursch et al. 2008). Pluripotent stem cells (PSCs) such as embryonic and induced pluripotent stem cells (ESCs, iPSCs) may represent a novel and potentially unlimited source of chondrocytes and tissues for therapeutic applications as these cells are able to generate a broad spectrum of cell types under appropriate conditions in vitro.
Chondrocytes develop from paraxial mesoderm that is induced in the early embryo in an ordered temporal pattern following the generation of lateral plate mesoderm (LPM) fated to give rise to hematopoietic and cardiovascular lineages (Lawson, Meneses et al. 1991, Kinder, Tsang et al. 1999). Following induction, strips of paraxial mesoderm are segmented into somites (Tam and Tan 1992, Kulesa and Fraser 2002). Somite development is regulated, in part, by the transcription factors paraxis (TCF15) and TBX18, whose expression coincides with induction of paraxial mesoderm (Burgess, Rawls et al. 1996, Bussen, Petry et al. 2004, Singh, Petry et al. 2005). Individual somites are then patterned into the ventral sclerotome, which forms the axial skeleton, including cartilage and the vertebral column, and the dorsal dermomyotome which develops into skeletal muscles and the dermis of the back (Hirsinger, Jouve et al. 2000). Specification of the sclerotome is marked by the expression of two transcription factors, Meox1 (Mankoo, Skuntz et al. 2003) and Nkx3.2 (Bapx1). A population of collagen 2 (Col2a1) positive mesenchymal cells with chondrogenic potential develops from sclerotome-derived cells at E12.5 of mouse development (Akiyama, Chaboissier et al. 2002, Dao, Jonason et al. 2012).
While methods for differentiating progenitor cells to the chondrogenic lineage are established, the ability to specify ACs, and ultimately stable cartilage tissue containing non-hypertrophic chondrocytes, remains poorly understood. ACs are derived from interzone cells, a fibrotic population of cells that forms at future sites of synovial joints, marked by the upregulation of Wnt9a/14 and growth and differentiation factor 5 (GDF5/BMP14), a member of the TGFβ superfamily (Archer, Dowthwaite et al. 2003, Pacifici, Koyama et al. 2006). Lineage tracing studies have shown that GDF5-expressing interzone cells give rise to several joint tissues including ACs, but do not contribute to the GPC population (Koyama, Shibukawa et al. 2008). GPCs, by contrast, develop from the condensing chondrogenic mesenchyme and express BMP 2, 4 and 7, as well as hypertrophy related genes including collagen 10. Distinct regions of ACs and GPCs are observed as early as postnatal day 7-8 when the secondary ossification center begins to form (Murakami, Balmes et al. 2004, Blumer, Longato et al. 2007). These observations suggest that ACs and GPCs are generated from separate progenitor populations during development and as such, may represent distinct lineages.
A number of studies have demonstrated that it is possible to derive chondrocytes from mouse (m) and human ESCs and iPSCs in vitro. Most, however, used serum-based media to support the early stages of differentiation resulting in the generation of mixed lineage end stage cultures (Kramer, Hegert et al. 2000, zur Nieden, Kempka et al. 2005, Hwang, Kim et al. 2006, Hwang, Varghese et al. 2008, Jukes, Both et al. 2008, Yamashita, Krawetz et al. 2008). Recent studies have reported the use of defined culture media with specific pathway agonists and antagonists to direct differentiation (Nakayama, Duryea et al. 2003, Darabi, Gehlbach et al. 2008, Tanaka, Jokubaitis et al. 2009). In mESCs, Tanaka et al (2009) showed that the combination of Wnt signaling with BMP inhibition resulted in the generation of paraxial mesoderm with chondrogenic potential, identified by the expression of PDGFRalpha and a lack of expression of Flk-1. This mesoderm also displayed some cardiac potential but showed no capacity to generate hematopoietic cells indicating that dependency on BMP signaling distinguishes different types of mesoderm.
Oldershaw et al. (Oldershaw, Baxter et al. 2010) used a serum free protocol. No tissues were obtained in vitro or in vivo with the method of Oldershaw.
Umeda et al (Umeda, Zhao et al. 2012) used a method using PDGF stimulation that produced nodules comprising Runx2 expressing cells.
Osteoarthritis is a degenerative disease that mainly affects the joint-lining articular cartilage of the joint. Articular cartilage has very limited capacity to regenerate itself upon injury, thus cell and tissue replacement strategies are the only means of replacing this tissue effectively. Methods of producing human cartilage from pluripotent stem cells are currently lacking, despite great need for such tissues for drug discovery and cartilage replacement strategies in patients with joint diseases such as osteoarthritis.